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CN-108513583-B - Recombinant maize B chromosome sequences and uses thereof

CN108513583BCN 108513583 BCN108513583 BCN 108513583BCN-108513583-B

Abstract

The present invention provides maize B chromosome genome loci and methods for agronomic practice.

Inventors

  • R kreistit
  • J.C. Lamb
  • T.S. rem
  • S-P.Yang
  • ZHOU XUEFENG

Assignees

  • 孟山都技术公司

Dates

Publication Date
20260508
Application Date
20160930
Priority Date
20151002

Claims (19)

  1. 1. A recombinant maize B chromosome comprising a target DNA inserted into a maize B chromosome sequence at a target locus having the sequence of SEQ ID No. 881, wherein the target DNA has the sequence of SEQ ID No. 904 on the right and the sequence of SEQ ID No. 903 on the left of the target DNA, and wherein the target DNA encodes a peptide or RNAi construct.
  2. 2. The recombinant maize B chromosome of claim 1, wherein the target DNA is integrated at a double strand break produced by one or more site-specific genome modification enzymes.
  3. 3. The recombinant maize B chromosome of claim 2, wherein the one or more site-specific genome modification enzymes are selected from endonucleases, recombinases, transposases, or any combination thereof.
  4. 4. The recombinant maize B chromosome of claim 2, wherein the one or more site-specific genome modification enzymes are endonucleases selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, arger nucleases, DNA-directed recombinases, DNA-directed endonucleases, RNA-directed recombinases and RNA-directed endonucleases.
  5. 5. The recombinant maize B chromosome of any of claims 1-4, wherein the target DNA comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group consisting of an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen utilization efficiency gene expression cassette, a moisture utilization efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, or a site-specific genomic modification enzyme gene expression cassette.
  6. 6. A method of making a transgenic maize cell comprising a target DNA integrated in the B chromosome: (a) Selecting at least one target site within the maize B chromosome sequence having the sequence of SEQ ID NO 881; (b) Selecting a site-specific genomic modification enzyme that specifically cleaves the target site; (c) Introducing the site-specific genome modification enzyme into the maize cell; (d) Introducing the target DNA into the maize cell; (e) Integrating the target DNA into the target site, and (F) Selecting a transgenic maize cell comprising said target DNA integrated into said B chromosome, wherein the right flank of said target DNA has the sequence of SEQ ID No. 904 and the left flank of said target DNA has the sequence of SEQ ID No. 903.
  7. 7. The method of claim 6, wherein the target DNA is integrated into the B chromosome by homology directed repair.
  8. 8. The method of claim 6, wherein the target DNA is integrated into the B chromosome by non-homologous end joining.
  9. 9. The method of any one of claims 6 to 8, wherein the site-specific genomic modification enzyme is selected from an endonuclease, a recombinase, a transposase, or any combination thereof.
  10. 10. The method of claim 9, wherein the site-specific genomic modification enzyme is an endonuclease selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, arginase, DNA-directed recombinases, DNA-directed endonucleases, RNA-directed recombinases, and RNA-directed endonucleases.
  11. 11. The method of claim 6, wherein the target DNA comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group consisting of an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen utilization efficiency gene expression cassette, a moisture utilization efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, or a site-specific genome modification enzyme gene expression cassette.
  12. 12. The recombinant maize B chromosome of claim 2, wherein the one or more site-specific genome modification enzymes are endonucleases selected from the group consisting of a type I CRISPR-Cas system, a type II CRISPR-Cas system, and a type III CRISPR-Cas system.
  13. 13. The recombinant maize B chromosome of claim 2, wherein the one or more site-specific genome modification enzymes are endonucleases selected from the group ：Cpf1、Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3 and Csf4 nucleases consisting of.
  14. 14. The recombinant maize B chromosome of claim 2, wherein the one or more site-specific genome modification enzymes are dCas 9-recombinase fusion proteins.
  15. 15. The recombinant maize B chromosome of claim 2, wherein the one or more site-specific genome modification enzymes are a tyrosine recombinase, a serine recombinase, a Cre recombinase, a Flp recombinase, a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase.
  16. 16. The method of claim 9, wherein the site-specific genome modification enzyme is an endonuclease selected from the group consisting of a type I CRISPR-Cas system, a type II CRISPR-Cas system, and a type III CRISPR-Cas system.
  17. 17. The method of claim 9, wherein the site-specific genome modification enzyme is an endonuclease selected from the group consisting of ：Cpf1、Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3 and Csf4 nucleases.
  18. 18. The method of claim 9, wherein the site-specific genome modification enzyme is dCas 9-recombinase fusion protein.
  19. 19. The method of claim 9, wherein the site-specific genome modification enzyme is a tyrosine recombinase, a serine recombinase, a Cre recombinase, a Flp recombinase, a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase.

Description

Recombinant maize B chromosome sequences and uses thereof Cross-reference to related applications and incorporation of sequence listing The present application claims priority from U.S. provisional patent application Ser. No. 62/236,709, filed on 10 month 02 of 2015, U.S. provisional patent application Ser. No. 62/237,048, filed on 10 month 05 of 2015, and U.S. provisional patent application Ser. No. 62/240,770, filed on 13 of 10 month 2015, which are incorporated herein by reference in their entirety. The sequence listing contained in the documents "P34350US00_seq.text" (3,202,544 bytes (measured in operating system MS Windows), created 10/02/2015, 10/02/2015 filed with U.S. provisional patent application No. 62/236,709), "P34350US01_seq.txt" (3,202,584 bytes (measured in operating system MS Windows), created 10/05/2015, 10/05, filed with U.S. provisional patent application No. 62/237,048), and "P34350US02_seq.txt" (3,655,665 bytes (measured in operating system MS Windows), created 10/13/2015, 10/13/2015 filed with U.S. provisional patent application No. 62/240,770) are incorporated herein by reference in their entirety. The computer readable form of the sequence listing is submitted with the present application by electronic submission and is incorporated herein by reference in its entirety. The sequence listing is contained in a file named 61653_ANNIV_ST25.Txt, which is 3,640,410 bytes in size (measured in operating system MS Windows) and was created at 2016, 9, 30. Background The B chromosome is an overdose chromosome that exists in many organisms, and differs from the standard nuclear chromosome (a chromosome) in that the former rarely carries an active gene. These chromosomes are not essential for life. The first transgenic plant was generated in the 90 s of the 20 th century as a result of random insertion of transgenic DNA into the nuclear A chromosome (e.g., roundupSoybean). Plant breeders and farmers desire crops with multiple traits as more transgenic events have been created that confer individual traits (e.g., insect resistance, drought tolerance, herbicide tolerance, quality traits). As the number of traits per plant increases, the resources and technical challenges required to produce a breeding complex of multiple traits present on the a chromosome in the elite germplasm correspondingly increase. In addition, there is a risk of linkage drag in the case of increased traits. In maize, trait compounding and a-chromosome linked burdensome risk reduction can be addressed by developing next generation transgenic traits on the B chromosome. Brief Description of Drawings FIG. 1 images of maize root tip cells with chromosomes stained with DAPI fluorescent stain. Arrows show 20B chromosomes in the nuclei of cells from individual plants. The magnification of the image is 40 times. FIG. 2 localization of maize B chromosome/A chromosome translocation using B chromosome primers. Control PCR amplicon regions are indicated BCHR004 for the proximal region, BCHR006 for the proximal euchromatin region, and BCHR007 for the distal region. The region of PCR amplification primers identified from the unique B chromosome sequences are shown as primers SEQ ID NO 889+890;SEQ ID NO:891+892, and SEQ ID NO 893+894. The abbreviations on the B chromosome schematic are CK for the B chromosome centromere region, PH for the proximal heterochromatin region, PE1/PE2 for the region of the proximal heterochromatin region, DH1-4 for the region of the distal heterochromatin region, and DE for the distal heterochromatin region. Summary of The Invention Several embodiments relate to a recombinant nucleic acid comprising a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to at least 0.25Kb、0.5Kb、0.75Kb、1Kb、1.25Kb、1.5Kb、1.75Kb、2Kb、2.25Kb、2.5Kb、2.75Kb、3Kb、3.25Kb、3.5Kb、3.75Kb、4Kb、4.25Kb、4.5Kb、4.75Kb Kb of a maize B chromosome sequence selected from the group consisting of SEQ ID NOS: 1-126 and 128-888, and at least one DNA of interest, wherein at least one of the DNA of interest is integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the DNA of interest is integrated at a double strand break generated by one or more site-specific genome modification enzymes. In some embodiments, the DNA of interest encodes a site-specific genomic modification enzyme. In some embodiments, the target DNA encodes one or more endonucleases, recombinases, transposases, helicases, or any combination thereof. In some embodiments, the target DNA comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group consisting of an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen utilization efficiency gene expression cassette, a moisture utilization efficiency gene expre